The internal circadian clock is a molecular time-keeping mechanism that generates a biological rhythm, regulating diverse physiological processes such as blood pressure, sleep-wake cycles and body temperature in mammals (Dunlap (1999) Cell 96:271–290; Cermakian, et al. (2000) Nature Reviews Molecular Cell Biology 1:59–67; King, et al. (2000) Annu. Rev. Neurosci. 23:713–742). Circadian clocks have been conserved throughout evolution and are present in almost all living organisms. The master pacemaker resides in the suprachiasmatic nucleus (SCN) in mammals (Reppert, et al. (1997) Cell 89:487–490). The SCN consists of multiple, single-cell circadian oscillators, which operate in a cell autonomous fashion. They are synchronized to fire rhythmically, generating a coordinated, circadian, rhythmic output in intact animals (Welsh, et al. (1995) Neuron 14:697–706; Liu, et al. (1997) Cell 91:855–860). Dawn and dusk coordinate or entrain the circadian clock through neural pathways connecting the retina to the SCN, so that the master clock and its output rhythms do not drift from a 24 hour cycle, but remain aligned with the solar day. Transient disruption of circadian timing following transmeridian flights leads to jet lag, and chronic alterations of the central clock mechanism of shift workers, which is approximately 25% of the working population, may contribute to poor health and sleep disorders. Moreover, specific rhythm defects may be involved in neuropsychiatric illnesses. Therefore, the need exists to develop mechanisms of regulating the circadian rhythm in humans.
Interacting positive and negative transcriptional-translational feedback loops drive circadian oscillations in both Drosophila and mammals. The best-characterized feedback loop in mice, involves the regulation of three Period genes (mPER1–3) and two Cryptochrome genes (mCRY1 and mCRY2)(Todo, et al. (1996) Science 272:109–112; Shearman, et al. (1997) Neuron 19:1261–1269; Sun, et al. (1997) Cell 90:1003–1011; Tei, et al. (1997) Nature 389:512–516; van der Horst, et al. (1999) Nature 398:627–630). The positive limb of this feedback loop requires the function of two basic helix-loop-helix-PAS (bHLH-PAS) proteins, CLOCK and BMAL1 (also known as MOP3) (King, et al. (1997) Cell 89:641–653; Gekakis, et al. (1998) Science 280:1564–1569; Hogenesch, et al. (1998) Proc. Natl. Acad. Sci. USA 95:5474–5479). It is believed that transcription of mPer and mCry is driven by accumulating CLOCK:BMAL heterodimers, which, in turn bind to consensus E-box elements (CACGTG) in their promoter regions (Darlington, et al. (1998) Science 280:1599–1603; Jin, et al. (1999) Cell 96:57–68). Heteromultimeric complexes formed from the products of the mPER and mCRY genes enter the nucleus, where the mCRY proteins shut off CLOCK:BMAL1-mediated transcription. At the same time, mPER2 increases levels of Bmal1 RNA through an as yet uncharacterized mechanism. This leads ultimately to renewal of BMAL1 levels, which increase CLOCK:BMAL1 heterodimers to drive mPer/mCRY transcription and restart the cycle (Kume, et al. (1999) Cell 98:193–205; Shearman, et al. (2000) Science 288:1013–1019). MOP4 (also termed NPAS2) is another member of the bHLH-PAS family of transcription factors and shares high homology at the amino acid level with CLOCK (Hogenesch, et al. (1997) J. Biol. Chem. 272:8581–8593; Zhou, et al. (1997) Proc. Natl. Acad. Sci. USA 94:713–718). In cultured cells, MOP4, like CLOCK, also functions optimally as a heterodimeric partner to BMAL1. The MOP4:BMAL1 heterodimer recognizes the same consensus E-box element as CLOCK:BMAL1 (Hogenesch, et al. (1998) Proc. Natl. Acad. Sci. USA 95:5474–5479), and CRY1 and CRY2 can inhibit MOP4:BMAL1-dependent E-box activation of genes such as Per1 and vasopressin (Kume, et al. (1999) Cell 98:193–205). However, the low level of MOP4 expression (Hogenesch, et al. (1998) Proc. Natl. Acad. Sci. USA 95:5474–5479) and the absence of mRNA cycling in the SCN (Shearman, et al. (1999) Neuroscience 89:387–397) has put into question its involvement in the core circadian feedback loop. Initially, it was believed that clock proteins were present only in specialized pacemaker neurons, such as those within the SCN. Recently, however, molecular clocks similar to those operating in SCN neurons have been uncovered in peripheral tissues (Zylka, et al. (1998) Neuron 20:1103–1110) and even in immortalized rat-1 fibroblast cell-lines (Balsalobre, et al. (1998) Cell 93:929–937). In peripheral tissues, such as the liver, kidney, and heart, circadian rhythms in RNA abundance are apparent for each of the mPER genes, although the phase of oscillation is delayed 3–9 hours relative to the oscillation in the SCN (Zylka, et al. (1998) Neuron 20:1103–1110). Clock gene oscillations are lost in SCN-lesioned animals (Sakamoto, et al. (1998) J. Biol. Chem. 273:27039–27042). Furthermore, gene oscillations dampen more rapidly in cultures of peripheral tissues than SCN cells in vitro, where they are sustained for weeks (Yamazaki, et al. (2000) Science 288:682–685). This suggests that the peripheral oscillations may be driven or synchronized by the SCN. It has been suggested that the SCN clock may synchronize peripheral clocks via both neural and hormonal signals (Ikonomov, et al. (1998) Prog. Neurobiol. 54:87–97; Ishida, et al. (1999) Proc. Natl. Acad. Sci. USA 96:8819–8820; Akashi, et al. (2000) Genes Dev. 14:645–649). Examples of stimuli that phase-shift central circadian oscillators include vasoactive intestinal peptide (Watanabe, et al. (2000) Brain Res. 877:361–366), delta opioid agonists (Byku, et al. (2000) Brain Res. 873:189–196), neuropeptide Y (Yannielli, et al. (2000) Neuroreport 11:1587–1591), and GABA (Liu, et al. (2000) Neuron 25:123–128). Steroid hormones and catecholamines are attractive candidate regulators of peripheral clocks and examples of hormonal phase-shifting of circadian genes in peripheral organs have begun to emerge (Balsalobre, et al. (2000) Science 289:2344–2347). Circulating concentrations of both steroids and catecholamines undergo circadian variability (Tronche, et al. (1998) Curr. Opin. Genet. Dev. 8:532–538; Czeisler, et al. (1999) Recent Prog. Horm. Res. 54:97–130; McCarty, et al. (1981) Physiol. Behav. 26:27–31; Muller (1999) Am. J. Hypertens. 12:35S-42S). While catecholamines can regulate gene expression via signaling cascades downstream of membrane receptors (Weiner and Molinoff (1995) Catecholamines. In: Basic Neurochemistry: Molecular Cellular and Medical Aspects, G. J. Siegel, ed. (New York, N.Y.; Raven press), pp. 276–312), steroid hormones function by activating nuclear hormone receptors (Perlmann, et al. (1997) Cell 90:391–397) which function as ligand-dependent transcription factors (Lin, et al. (1998) Cold Spring Harb. Symp. Quant. Biol. 63:577–585).
Though there is no molecular data, several lines of evidence suggest that a vascular clock exists. For example, blood pressure undergoes a marked circadian variability (Millar-Craig, et al. (1978) Lancet 1:795–797; Panza, et al. (1991) N. Engl. J. Med. 325:986–990), which is increased in patients with hypertension (Lemmer (1999) Acta Physiol. Pharmacol. Bulg. 24:71–80) and coincides with a temporal variability in the incidence of acute vascular events, such as myocardial infarction, sudden cardiac death and stroke (Marshall (1977) Stroke 8:230–231; Tsementzis, et al. (1985) Neurosurgery 17:901–904; Ridker, et al. (1990) Circulation 82:897–902). Evidence also suggests that endothelial function has a circadian variation with attenuated activity in the morning (Elherik, et al. (2000) Circulation 102(18):902 Suppl. S). Furthermore, previous studies have shown a circadian variability in the local pressor response to infused catecholamines in humans (Hossmann, et al. (1980) Cardiovasc. Res. 14:125–129).
Previous observations have hinted at a circadian role for retinoid nuclear receptors and vitamin A. For example, targeted gene disruption of the retinoid-related orphan receptor, RORβ, extends the period length of the free-running activity rhythm in mice and mildly affects circadian rhythmicity (Andre, et al. (1998) EMBO J. 17:3867–3877). Similarly, vitamin A deficiency reduces both the expression of AA-NAT mRNA and melatonin content in the pineal gland (Fu, et al. (1999) J. Pineal. Res 27:34–41). The majority of retinol circulates bound to a 21 kDa retinol-binding protein (RBP) (Soprano, et al. (1994) In: The Retinoids, Biology, Chemistry, and Medicine, M. B. Sporn, A. B. Roberts and D. S. Goodman, eds. (New York, N.Y.: Raven Press), pp 257–282), which reportedly undergoes diurnal variation in humans (Hongo, et al. (1993) J. Nutr. Sci. Vitaminol. (Tokyo) 39:33–46). Plasma retinol is internalized by cells from RBP through a process involving the action of a number of cellular retinol binding proteins (CRBPs) including the interphotoreceptor retinol binding protein (IRBP), which has been shown to under circadian variation in zebrafish (Rajendran, et al (1996) J. Exp. Biol 199:2775–2787). Interestingly, RBP is a member of the lipcalin protein family, as is prostaglandlin D2 synthase (PGDS) (Flower (1996) Biochem. J. 318:1–14). Circadian rhythmicity in PGDS expression and consequent biosynthesis of PGD2 is thought to be relevant to regulation of the sleep-wake cycle (Pinzar, et al. (2000) Proc. Natl. Acad. Sci. USA 97:4903–4907). In addition, lipocalin protein family resembles the Drosophila takeout (TO) gene superfamily, at least one member of which is controlled by the clock and affects feeding behaviour (Sarov-Blat, et al. (2000) Cell 101:647–656); So, et al. (2000) Mol. Cell Biol. 20:6935–6944). Some of these observations indicate a role for retinoids, not only in peripheral circadian physiology, but also in functions that may be directly controlled by the brain, however, a molecular mechanism for retinoid action is not known.
Methods of modulating or screening for compounds that modulate the circadian rhythm have focused on CLOCK (U.S. Pat. No. 6,057,125 to Takahaski, et al.), the CLOCK:BMAL1 interaction (PCT Publication WO 99/57137), CRY and PER2 proteins (PCT Publication WO 01/07654), human and mouse PER2 proteins (PCT Publication WO 99/14324), and MOP4 (PCT Publication WO 99/28464). Similarly, modulation of neuropeptide Y Y5 receptor ligand (PCT publication WO 99/05911) and neurokinin-1 receptor antagonist (U.S. Pat. No. 6,274,604 to Mendel) are provided as a means of regulating circadian rhythm.
The present invention provides a method of regulating circadian rhythm by modulating the peripheral clock components in the vasculature by administering retinoid nuclear receptor ligands.